WO2002036815A2 - Analyse genetique de micro-organismes - Google Patents

Analyse genetique de micro-organismes Download PDF

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Publication number
WO2002036815A2
WO2002036815A2 PCT/GB2001/004760 GB0104760W WO0236815A2 WO 2002036815 A2 WO2002036815 A2 WO 2002036815A2 GB 0104760 W GB0104760 W GB 0104760W WO 0236815 A2 WO0236815 A2 WO 0236815A2
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WO
WIPO (PCT)
Prior art keywords
gene
taxonomic
amplification
product
primers
Prior art date
Application number
PCT/GB2001/004760
Other languages
English (en)
Other versions
WO2002036815A3 (fr
Inventor
Stephen Minter
James Ivor Prosser
Carol Jane Phillips
Original Assignee
Ncimb Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ncimb Ltd. filed Critical Ncimb Ltd.
Priority to AU2002214126A priority Critical patent/AU2002214126A1/en
Priority to GB0314840A priority patent/GB2390159A/en
Publication of WO2002036815A2 publication Critical patent/WO2002036815A2/fr
Publication of WO2002036815A3 publication Critical patent/WO2002036815A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the method according to the present invention has the advantage that it may be carried out irrespective of the need to culture and purify the microorganisms.
  • uncultured organisms 99-99.99% of the total population
  • the present invention is of considerable commercial importance. This applies both to uncultured members of established groups and to the major, novel and uncharacterised microorganisms discovered through molecular analysis.
  • Figure 2 illustrates the amplification and linkage of a taxonomic gene fragment and a functional gene fragment wherein linkage of the amplified gene fragments is achieved through the hybridisation of mutually complementary regions of DNA;
  • Figure 1 shows a section of genomic DNA from a microbial sample, showing a taxonomic gene remote from a functional gene.
  • Numbers 1 and 2 represent 16S rDNA taxonomic gene specific primers, which could be either general or specific, and numbers 3 and 4 represent primers for the functional gene, which may be specific to a gene of interest or may be random.
  • Figure 1(d) shows the repair of single stranded breaks by Klenow fragment (3 '-5').
  • Figure 2(b) shows an amplification product 11 of gene 5 using primer pair 7 and 8 , product 11 comprising DNA strands 12 and 13. Also shown is an amplification product 14 of gene 6 using primer pair 9 and 10 , product 14 comprising DNA strands
  • Figure 2(c) shows that upon denaturating of products 11 and 14 DNA strands 12 and
  • DNA polymerase is then able to initiate polymerisation from this hybridised region to produce strands complementary to the gene-derived sequences of DNA strands 13 and 16 (as illustrated by the broken arrows).
  • a first broth culture of C. albicans was set up by inoculating 10 ml of YPD broth with a single C. albicans colony. The broth was incubated at 30°C with shaking overnight. To ensure that the cells to be used were in the exponential phase of growth, during which they are most active, 500 ⁇ l of the first broth culture was taken to inoculate 10 ml of fresh YPD medium and incubated at 30°C for a further 3 h to establish a second broth culture.
  • the cells of this second broth culture were then pelleted by centrifugation at 5,000 rpm for 5 min.
  • the cells were washed three times by re-suspending the cells in 1 ml PBS by pipetting and then centrifuging the cells at 5,000 rpm for 2 min.
  • the cells were re-suspended in 460 ⁇ l of buffer A [0.1 M potassium phosphate buffer (pH 7.5), 1.2 M sorbitol]. An aliquot of these cells was retained for use as a control (representing non-permeabilised cells). The remaining cells were permeabilised by incubation with 40 ⁇ l zymolyase and 1 ⁇ l ⁇ - mercaptoethanol at 37°C for 15 min with gentle agitation. The cells were then washed again three times with PBS as above. The final cell pellet was resuspended in 100 ⁇ l of PBS.
  • the taxonomic and functional genes selected for amplification and hybridisation were the 18S rRNA gene and the chitin synthase gene respectively.
  • the primers pairs used to achieve amplification and linking of the genes were nu-SSU-0817 and nu-SSU- 1536TAIL, which are specific for the 18S rRNA gene, and CHS8-8TAIL and CHS8- 11, which are specific for the chitin synthase gene (details of the primers are included in Table 1).
  • nu-SSU-1536TAIL and CHS8-8TAIL both contain, in addition to sequences complementary to their respective target genes, "tail" sequences that complement one another. In situ PCR reactions were set up such that each 50 ⁇ l reaction volume contained:
  • nu-SSU-1536TAlL and CHS8-8TAIL primers 0.2 pmol of nu-SSU-1536TAlL and CHS8-8TAIL primers.
  • PCR amplifications were carried out on a Hybaid Omni-E Thermal Cycler (Hybaid) as follows: 94°C for 5 min (one cycle); 94°C for 40 s, 55°C for 2 min, 74°C for 1 min (10 cycles); 94°C for 40 s, 55°C for 40 s, 74°C for 1 min (23 cycles) followed by a final incubation at 74°C for 7 min.
  • Hybaid Hybaid Omni-E Thermal Cycler
  • the cells were pelleted by centrifugation at 5,000 rpm for 2 min. The supernatant was removed and retained for analysis. The cells were then washed three times with 200 ⁇ l PBS as described above. Following the third centrifugation, the supernatant wash solution was retained for analysis. The final cell pellet was resuspended in 30 ⁇ l PBS and incubated at 95°C for 5 min to lyse the cells. The original supernatant from the PCR, the wash solution, and the lysed cell solution were then analysed by electrophoresis in a 1% (w/v) ethidium bromide stained agarose gel.
  • reaction products are present in lanes 1 to 6.
  • the reaction products contain the hybridisation product, comprising fragments of the 18S rRNA gene and the chitin synthase gene linked by the hybridised "tail" sequences (band indicated with arrow), 18S rRNA product and chitin synthase product. Excision and sequence analysis of the hybrid product band has confirmed its identity through the presence of fragments of both target genes.
  • the lane labelled M contains molecular weight markers, and the lane labelled C represents a negative control.
  • PCR product was centrifuged to separate the cells. The supernatant was analysed for PCR amplification products. The cells were washed with PBS, lysed and then analysed for PCR amplification products. Presence of the linked product within the PCR amplification products was confirmed by electrophoresis in a 1% (w/v) ethidium bromide stained agarose gel.
  • Bacterial cells may be separated from soil particles by dispersion in a Waring blender with 100 mM sodium phosphate buffer (pH 7.0) for 1 min. Coarse particles may then be allowed to settle for 1 min before the resultant suspension is serially diluted to remove the remaining soil particles. Bacterial cells may then be attached to paramagnetic beads by antibodies specific to bacterial cell wall proteins and the beads washed to remove any residual traces of soil particles, which could interfere with further processing. The beads may then be serially diluted in a microtitre plate format to give approximately one cell per well.
  • a gentle cell lysis solution 0.5 mg ml "1 for 30 min at room temperature
  • the bacterial wall can be permeabilised such that primers and reagents are able to enter the cell but nuclear material is not able to escape.
  • a first round of PCR using primers 1, 2, 3 and 4 (Table 4), can then be carried out to create amplicons of both the 16S rRNA and amoA genes (cycling conditions - 10 cycles of 30 s at 94°C, 60 s at 55°C, and 45 s at 72°C).
  • a conventional PCR (cycling conditions - 60 s at 94°C followed by 30 cycles of 30 s at 94°C, 60 s at 55°C, 45 s at 72°C, followed by 5 min at 72°C) may be carried out using the primer set 1 (16S rRNA for ammonia oxidiser) and 4 (amoA for ammonia oxidiser).
  • the amplified product can then be sequenced using internal sequencing primers.
  • Oligonucleotide probe sequences suitable for amplification and linkage of phylogenetic and functional genes in environmental samples.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédé d'analyse génétique d'un spécimen microbien consistant à relier des caractéristiques taxinomiques à des caractéristiques fonctionnelles afin d'exécuter une amplification conjointe d'un gène taxinomique et d'un gène fonctionnel de spécimen biologique, puis d'effectuer la liaison des deux produits amplifiés. On peut ensuite analyser le produit de liaison.
PCT/GB2001/004760 2000-10-28 2001-10-29 Analyse genetique de micro-organismes WO2002036815A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002214126A AU2002214126A1 (en) 2000-10-28 2001-10-29 Genetic analysis of microorganisms
GB0314840A GB2390159A (en) 2000-10-28 2001-10-29 Genetic analysis of microorganisms

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0026424.2 2000-10-28
GBGB0026424.2A GB0026424D0 (en) 2000-10-28 2000-10-28 Genetic analysis of microorganisms

Publications (2)

Publication Number Publication Date
WO2002036815A2 true WO2002036815A2 (fr) 2002-05-10
WO2002036815A3 WO2002036815A3 (fr) 2003-10-16

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AU (1) AU2002214126A1 (fr)
GB (2) GB0026424D0 (fr)
WO (1) WO2002036815A2 (fr)

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2492352A1 (fr) * 2009-10-22 2012-08-29 Biotools Biotechnological & Medical Laboratories, S.A. Composition, méthode et trousse pour la détection de bactéries par séquençage
US20160201125A1 (en) * 2011-02-18 2016-07-14 Raindance Technologies, Inc. Compositions and methods for molecular labeling
US9840732B2 (en) 2012-05-21 2017-12-12 Fluidigm Corporation Single-particle analysis of particle populations
US10960397B2 (en) 2007-04-19 2021-03-30 President And Fellows Of Harvard College Manipulation of fluids, fluid components and reactions in microfluidic systems
US11077415B2 (en) 2011-02-11 2021-08-03 Bio-Rad Laboratories, Inc. Methods for forming mixed droplets
US11174509B2 (en) 2013-12-12 2021-11-16 Bio-Rad Laboratories, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample
US11187702B2 (en) 2003-03-14 2021-11-30 Bio-Rad Laboratories, Inc. Enzyme quantification
US11254968B2 (en) 2010-02-12 2022-02-22 Bio-Rad Laboratories, Inc. Digital analyte analysis
US11351510B2 (en) 2006-05-11 2022-06-07 Bio-Rad Laboratories, Inc. Microfluidic devices
US11390917B2 (en) 2010-02-12 2022-07-19 Bio-Rad Laboratories, Inc. Digital analyte analysis
US11511242B2 (en) 2008-07-18 2022-11-29 Bio-Rad Laboratories, Inc. Droplet libraries
US11635427B2 (en) 2010-09-30 2023-04-25 Bio-Rad Laboratories, Inc. Sandwich assays in droplets
US11786872B2 (en) 2004-10-08 2023-10-17 United Kingdom Research And Innovation Vitro evolution in microfluidic systems
US11819849B2 (en) 2007-02-06 2023-11-21 Brandeis University Manipulation of fluids and reactions in microfluidic systems
US11898193B2 (en) 2011-07-20 2024-02-13 Bio-Rad Laboratories, Inc. Manipulating droplet size
US11901041B2 (en) 2013-10-04 2024-02-13 Bio-Rad Laboratories, Inc. Digital analysis of nucleic acid modification

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EP0628640A1 (fr) * 1993-06-04 1994-12-14 Becton, Dickinson and Company Amplification simultanée de cibles multiples
WO1998038298A1 (fr) * 1997-02-27 1998-09-03 Gesher - Israel Advanced Biotecs (1996) Ltd. Procede de reunion de plusieurs fragments d'adn

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COTTRELL MATTHEW T ET AL: "Selected chitinase genes in cultured and uncultured marine bacteria in the alpha- and gamma-subclasses of the proteobacteria." APPLIED AND ENVIRONMENTAL MICROBIOLOGY., vol. 66, no. 3, March 2000 (2000-03), pages 1195-1201, XP002241674 ISSN: 0099-2240 *
HODSON ROBERT E ET AL: "In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities." APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 61, no. 11, 1995, pages 4074-4082, XP001146824 ISSN: 0099-2240 cited in the application *
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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11187702B2 (en) 2003-03-14 2021-11-30 Bio-Rad Laboratories, Inc. Enzyme quantification
US11786872B2 (en) 2004-10-08 2023-10-17 United Kingdom Research And Innovation Vitro evolution in microfluidic systems
US11351510B2 (en) 2006-05-11 2022-06-07 Bio-Rad Laboratories, Inc. Microfluidic devices
US11819849B2 (en) 2007-02-06 2023-11-21 Brandeis University Manipulation of fluids and reactions in microfluidic systems
US10960397B2 (en) 2007-04-19 2021-03-30 President And Fellows Of Harvard College Manipulation of fluids, fluid components and reactions in microfluidic systems
US11618024B2 (en) 2007-04-19 2023-04-04 President And Fellows Of Harvard College Manipulation of fluids, fluid components and reactions in microfluidic systems
US11224876B2 (en) 2007-04-19 2022-01-18 Brandeis University Manipulation of fluids, fluid components and reactions in microfluidic systems
US11511242B2 (en) 2008-07-18 2022-11-29 Bio-Rad Laboratories, Inc. Droplet libraries
US11596908B2 (en) 2008-07-18 2023-03-07 Bio-Rad Laboratories, Inc. Droplet libraries
US11534727B2 (en) 2008-07-18 2022-12-27 Bio-Rad Laboratories, Inc. Droplet libraries
EP2492352A1 (fr) * 2009-10-22 2012-08-29 Biotools Biotechnological & Medical Laboratories, S.A. Composition, méthode et trousse pour la détection de bactéries par séquençage
EP2492352A4 (fr) * 2009-10-22 2013-03-20 Biotools Biotechnological & Medical Lab S A Composition, méthode et trousse pour la détection de bactéries par séquençage
US11254968B2 (en) 2010-02-12 2022-02-22 Bio-Rad Laboratories, Inc. Digital analyte analysis
US11390917B2 (en) 2010-02-12 2022-07-19 Bio-Rad Laboratories, Inc. Digital analyte analysis
US11635427B2 (en) 2010-09-30 2023-04-25 Bio-Rad Laboratories, Inc. Sandwich assays in droplets
US11077415B2 (en) 2011-02-11 2021-08-03 Bio-Rad Laboratories, Inc. Methods for forming mixed droplets
US11168353B2 (en) 2011-02-18 2021-11-09 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US10584332B2 (en) * 2011-02-18 2020-03-10 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US11747327B2 (en) 2011-02-18 2023-09-05 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US11768198B2 (en) 2011-02-18 2023-09-26 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US20160201125A1 (en) * 2011-02-18 2016-07-14 Raindance Technologies, Inc. Compositions and methods for molecular labeling
US11965877B2 (en) 2011-02-18 2024-04-23 Bio-Rad Laboratories, Inc. Compositions and methods for molecular labeling
US11754499B2 (en) 2011-06-02 2023-09-12 Bio-Rad Laboratories, Inc. Enzyme quantification
US11898193B2 (en) 2011-07-20 2024-02-13 Bio-Rad Laboratories, Inc. Manipulating droplet size
US9840732B2 (en) 2012-05-21 2017-12-12 Fluidigm Corporation Single-particle analysis of particle populations
US11901041B2 (en) 2013-10-04 2024-02-13 Bio-Rad Laboratories, Inc. Digital analysis of nucleic acid modification
US11174509B2 (en) 2013-12-12 2021-11-16 Bio-Rad Laboratories, Inc. Distinguishing rare variations in a nucleic acid sequence from a sample

Also Published As

Publication number Publication date
WO2002036815A3 (fr) 2003-10-16
GB2390159A (en) 2003-12-31
GB0314840D0 (en) 2003-07-30
AU2002214126A1 (en) 2002-05-15
GB0026424D0 (en) 2000-12-13

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